Novel In Vitro Platform for Studying the Cell Response to Healthy and Diseased Tendon Matrices.

Current in vitro models poorly represent the healthy or diseased tendon microenvironment, limiting the translation of the findings to clinics. The present work aims to establish a physiologically relevant in vitro tendon platform that mimics biophysical aspects of a healthy and tendinopathic tendon matrix using a decellularized bovine tendon and to characterize tendon cells cultured using this platform. Bovine tendons were subjected to various decellularization techniques, with the efficacy of decellularization determined histologically. The biomechanical and architectural properties of the decellularized tendons were characterized using an atomic force microscope. Tendinopathy-mimicking matrices were prepared by treating the decellularized tendons with collagenase for 3 h or collagenase-chondroitinase (CC) for 1 h. The tendon tissue collected from healthy and tendinopathic patients was characterized using an atomic force microscope and compared to that of decellularized matrices. Healthy human tendon-derived cells (hTDCs) from the hamstring tendon were cultured on the decellularized matrices for 24 or 48 h, with cell morphology characterized using f-actin staining and gene expression characterized using real-time PCR. Tendon matrices prepared by freeze-thawing and 48 h nuclease treatment were fully decellularized, and the aligned structure and tendon stiffness (1.46 MPa) were maintained. Collagenase treatment prepared matrices with a disorganized architecture and reduced stiffness (0.75 MPa), mimicking chronic tendinopathy. Treatment with CC prepared matrices with a disorganized architecture without altering stiffness, mimicking early tendinopathy (1.52 MPa). hTDCs on a healthy tendon matrix were elongated, and the scleraxis (SCX) expression was maintained. On tendinopathic matrices, hTDCs had altered morphological characteristics and lower SCX expression. The expression of genes related to actin polymerization, matrix degradation and remodeling, and immune cell invasion were higher in hTDCs on tendinopathic matrices. Overall, the present study developed a physiological in vitro system to mimic healthy tendons and early and late tendinopathy, and it can be used to better understand tendon cell characteristics in healthy and diseased states.

Combined Growth Factor Hydrogel Enhances Rotator Cuff Enthesis Healing in Rat But Not Sheep Model.

We hypothesized that a combined growth factor hydrogel would improve chronic rotator cuff tear healing in a rat and sheep model. Insulin-like growth factor 1, transforming growth factor ß1, and parathyroid hormone were combined into a tyraminated poly-vinyl-alcohol (PVA-Tyr) hydrogel and applied directly at the enthesis. In total, 30 Sprague-Dawley rats and 16 Romney ewes underwent unilateral rotator cuff tenotomy and then delayed repairs were performed after 3-4 weeks. The animals were divided into a control group (repair alone) and treatment group. The rotator cuffs were harvested at 12 weeks after surgery for biomechanical and histological analyses of the repair site. In the rat model, the stress at failure and Young's modulus were higher in the treatment group in comparison with the control group (73% improvement, p = 0.010 and 56% improvement, p = 0.028, respectively). Histologically, the repaired entheses in the treatment group demonstrated improved healing with higher semi-quantitative scores (10.1 vs. 6.55 of 15, p = 0.032). In the large animal model, there was no observable treatment effect. This PVA-Tyr bound growth factor system holds promise for improving rotator cuff healing. However, our approach was not scalable from a small to a large animal model. Further tailoring of this growth factor delivery system is still required. Level of Evidence: Basic Science Study; Biomechanics and Histology; Animal Model Impact Statement Previous studies using single-growth factor treatment to improve enthesis healing after rotator cuff repair have reported promising, but inconsistent results. A novel approach is to combine multiple growth factors using controlled-release hydrogels that mimic the normal healing process. In this study, we report that a combined growth factor hydrogel can improve the histological quality and strength of rotator cuff repair in a rat chronic tear model. This novel hydrogel growth factor treatment has the potential to be used in human clinical applications to improve healing after rotator cuff repair.

Ethnicity, sex, and socioeconomic disparities in the treatment of traumatic rotator cuff injuries in Aotearoa/New Zealand.

HYPOTHESIS AND BACKGROUND: Traumatic rotator cuff injuries can be a leading cause of prolonged shoulder pain and disability and contribute to significant morbidity and health care costs. Previous studies have shown evidence of sociodemographic disparities with these injuries. The purpose of this nationwide study was to better understand these disparities based on ethnicity, sex, and socioeconomic status, in order to inform future health care strategies. METHODS: Accident Compensation Corporation (ACC) is a no-fault comprehensive compensation scheme encompassing all of Aotearoa/New Zealand (population in 2018, 4.7 million). Using the ACC database, traumatic rotator cuff injuries were identified between January 2010 and December 2018. Injuries were categorized by sex, ethnicity, age, and socioeconomic deprivation index of the claimant. RESULTS: During the 9-year study period, there were 351,554 claims accepted for traumatic rotator cuff injury, which totaled more than NZ$960 million. The greatest proportion of costs was spent on vocational support (49.8%), then surgery (26.3%), rehabilitation (13.1%), radiology (8.1%), general practitioner (1.6%), and "Other" (1.1%). Asian, Maori (indigenous New Zealanders), and Pacific peoples were under-represented in the age-standardized proportion of total claims and had lower rates of surgery than Europeans. Maori had higher proportion of costs spent on vocational support and lower proportions spent on radiology, rehabilitation, and surgery than Europeans. Males had higher number and costs of claims and were more likely to have surgery than females. There were considerably fewer claims from areas of high socioeconomic deprivation. DISCUSSION AND CONCLUSION: This large nationwide study demonstrates the important and growing economic burden of rotator cuff injuries. Indirect costs, such as vocational supports, are a major contributor to the cost, suggesting improving treatment and rehabilitation protocols would have the greatest economic impact. This study has also identified sociodemographic disparities that need to be addressed in order to achieve equity in health outcomes.

Cytotoxicity of tranexamic acid to tendon and bone in vitro: Is there a safe dosage?

INTRODUCTION: Tranexamic acid (TXA) has been shown to be effective at reducing peri-operative blood loss and haemarthrosis in arthroplasty and arthroscopic soft tissue reconstructions. Intra-articular application, as an injection or peri-articular wash, is becoming increasingly common. Recent studies have shown TXA has the potential to be cytotoxic to cartilage, but its effects on human tendon and bone remain poorly understood. The aim of this study was to investigate whether TXA has any detrimental effects on tendon-derived cells and osteoblast-like cells and determine whether there is a safe dosage for clinical application. MATERIALS AND METHODS: Primary tendon-derived cells and osteoblast-like cells were harvested from hamstring tendons and trabecular bone explants, respectively, and analysed in vitro with a range of TXA concentrations (0 to 100 mg/ml) at time points: 3 and 24 h. The in vitro toxic effect of TXA was investigated using viability assays (alamarBlue), functional assays (collagen deposition), fluorescent microscopy and live/apoptosis/necrosis staining for cell death mechanisms in 2D monolayer and 3D collagen gel cell culture. RESULTS: There was a significant (P < 0.05) decrease in tendon-derived cell and osteoblast-like cell numbers following treatment with TXA ≥ 50 mg/ml after 3 h and ≥ 20 mg/ml after 24 h. In tendon-derived cells, increasing concentrations > 35 mg/ml resulted in significantly (P < 0.05) reduced collagen deposition. Fluorescence imaging confirmed atypical cellular morphologies with increasing TXA concentrations and reduced cell numbers. The mechanism of cell death was demonstrated to be occurring through apoptosis. CONCLUSIONS: Topical TXA treatment demonstrated dose- and time-dependent cytotoxicity to tendon-derived cells and osteoblast-like cells with concentrations 20 mg/ml and above in isolated 2D and 3D in vitro culture. On the basis of these findings, concentrations of less than 20 mg/ml are expected to be safe. Orthopaedic surgeons should show caution when considering topical TXA treatments, particularly in soft tissue and un-cemented arthroplasty procedures.

Changes in Physiological Tendon Substrate Stiffness Have Moderate Effects on Tendon-Derived Cell Growth and Immune Cell Activation.

Tendinopathy is characterised by pathological changes in tendon matrix composition, architecture, and stiffness, alterations in tendon resident cell characteristics, and fibrosis, with inflammation also emerging as an important factor in tendinopathy progression. The sequence of pathological changes in tendinopathy and the cellular effects of the deteriorating matrix are largely unknown. This study investigated the effects of substrate stiffness on tendon-derived cells (TDCs) and THP-1 macrophages using PDMS substrates representing physiological tendon stiffness (1.88 MPa), a stiff gel (3.17 MPa) and a soft gel (0.61 MPa). Human TDCs were cultured on the different gel substrates and on tissue culture plastic. Cell growth was determined by alamarBlue™ assay, cell morphology was analysed in f-actin labelled cells, and phenotypic markers were analysed by real-time PCR. We found that in comparison to TDCs growing on gels with physiological stiffness, cell growth increased on soft gels at 48 h (23%, p = 0.003). Cell morphology was similar on all three gels. SCX expression was slightly reduced on the soft gels (1.4-fold lower, p = 0.026) and COL1A1 expression increased on the stiff gels (2.2-fold, p = 0.041). Culturing THP-1 macrophages on soft gels induced increased expression of IL1B (2-fold, p = 0.018), and IL8 expression was inhibited on the stiffer gels (1.9-fold, p = 0.012). We also found that culturing TDCs on plastic increased cell growth, altered cell morphology, and inhibited the expression of SCX, SOX9, MMP3, and COL3. We conclude that TDCs and macrophages respond to changes in matrix stiffness. The magnitude of responses measured in TDCs were minor on the range of substrate stiffness tested by the gels. Changes in THP-1 macrophages suggested a more inflammatory phenotype on substrates with non-physiological stiffness. Although cell response to subtle variations in matrix stiffness was moderate, it is possible that these alterations may contribute to the onset and progression of tendinopathy.

A high-fat diet has negative effects on tendon resident cells in an in vivo rat model.

BACKGROUND: Tendinopathy is a major complication of diet-induced obesity. However, the effects of a high-fat diet (HFD) on tendon have not been well characterised. We aimed to determine: [1] the impact of a HFD on tendon properties and gene expression; and [2] whether dietary transition to a control diet (CD) could restore normal tendon health. METHODS: Sprague-Dawley rats were randomised into three groups from weaning and fed either a: CD, HFD or HFD for 12 weeks and then CD thereafter (HF-CD). Biomechanical, histological and structural evaluation of the Achilles tendon was performed at 17 and 27 weeks of age. Tail tenocytes were isolated with growth rate and collagen production determined. Tenocytes and activated THP-1 cells were exposed to conditioned media (CM) of visceral adipose tissue explants, and gene expression was analysed. RESULTS: There were no differences in the biomechanical, histological or structural tendon properties between groups. However, tenocyte growth and collagen production were increased in the HFD group at 27 weeks. There was lower SOX-9 expression in the HFD and HF-CD groups at 17 weeks and higher expression of collagen-Iα1 and matrix metalloproteinase-13 in the HFD group at 27 weeks. THP-1 cells exposed to adipose tissue CM from animals fed a HFD or HF-CD had lower expression of Il-10 and higher expression of Il-1ß. CONCLUSIONS: In this rodent model, a HFD negatively altered tendon cell characteristics. Dietary intervention restored some gene expression changes; however, adipose tissue secretions from the HF-CD group promoted an increased inflammatory state in macrophages. These changes may predispose tendon to injury and adverse events later in life.

Obesity Impairs Enthesis Healing After Rotator Cuff Repair in a Rat Model.

BACKGROUND: Being overweight or obese is associated with poor outcomes and an increased risk of failure after rotator cuff (RC) surgery. However, the effect of obesity on enthesis healing has not been well characterized. HYPOTHESES: Diet-induced obesity (DIO) would result in inferior enthesis healing in a rat model of RC repair, and a dietary intervention in the perioperative period would improve enthesis healing. STUDY DESIGN: Controlled laboratory study. METHODS: Male Sprague-Dawley rats were divided into 3 weight-matched groups (n = 26 per group): control diet (CD), high-fat diet (HFD), or HFD until surgery and then CD thereafter (HF-CD). After 12 weeks, the left supraspinatus tendon was detached, followed by immediate repair. Animals were sacrificed, and RCs were harvested at 2 and 12 weeks after surgery for biomechanical and histological evaluations. Metabolic end points were assessed using dual-energy X-ray absorptiometry and plasma analyses. RESULTS: DIO was established in the HFD and HF-CD groups before surgery and subsequently reversed in the HF-CD group after surgery. At 12 weeks after surgery, the body fat percentage (P = .0021) and plasma leptin concentration (P = .0025) were higher in the HFD group compared with the CD group. Histologically, the appearance of the repaired entheses was poorer in both the HFD and HF-CD groups compared with the CD group at 12 weeks after surgery, with semiquantitative scores of 6.20 (P = .0078), 4.98 (P = .0003), and 8.68 of 15, respectively. The repaired entheses in the HF-CD group had a significantly lower load to failure (P = .0278) at 12 weeks after surgery compared with the CD group, while the load to failure in the HFD group was low but not significantly different (P = .0960). There were no differences in the biomechanical and histological results between the groups at 2 weeks after surgery. Body mass at the time of surgery, plasma leptin concentration, and body fat percentage were negatively correlated with histology scores and plasma leptin concentration was correlated with load to failure at 12 weeks after surgery. CONCLUSION: DIO impaired enthesis healing in this rat RC repair model, with inferior biomechanical and histological outcomes. Restoring a normal weight with dietary changes after surgery did not improve healing outcomes. CLINICAL RELEVANCE: Obesity is a potentially modifiable factor that impairs RC healing and increases the risk of failure after surgery. Exploring interventions that improve the metabolic state of obese patients and counseling patients appropriately about their modest expectations after repair should be considered.

A Maternal High Fat Diet Leads to Sex-Specific Programming of Mechanical Properties in Supraspinatus Tendons of Adult Rat Offspring.

Background: Over half of women of reproductive age are now overweight or obese. The impact of maternal high-fat diet (HFD) is emerging as an important factor in the development and health of musculoskeletal tissues in offspring, however there is a paucity of evidence examining its effects on tendon. Alterations in the early life environment during critical periods of tendon growth therefore have the potential to influence tendon health that cross the lifespan. We hypothesised that a maternal HFD would alter biomechanical, morphological and gene expression profiles of adult offspring rotator cuff tendon. Materials and Methods: Female Sprague-Dawley rats were randomly assigned to either: control diet (CD; 10% kcal or 43 mg/g from fat) or HFD (45% kcal or 235 mg/g from fat) 14 days prior to mating and throughout pregnancy and lactation. Eight female and male offspring from each maternal diet group were weaned onto a standard chow diet and then culled at postnatal day 100 for tissue collection. Supraspinatus tendons were used for mechanical testing and histological assessment (cellularity, fibre organisation, nuclei shape) and tail tendons were collected for gene expression analysis. Results: A maternal HFD increased the elasticity (Young's Modulus) in the supraspinatus tendon of male offspring. Female offspring tendon biomechanical properties were not affected by maternal HFD. Gene expression of SCX and COL1A1 were reduced in male and female offspring of maternal HFD, respectively. Despite this, tendon histological organisation were similar between maternal diet groups in both sexes. Conclusion: An obesogenic diet during pregnancy increased tendon elasticity in male, but not female, offspring. This is the first study to demonstrate that maternal diet can modulate the biomechanical properties of offspring tendon. A maternal HFD may be an important factor in regulating adult offspring tendon homeostasis that may predispose offspring to developing tendinopathies and adverse tendon outcomes in later life.

Collagen-I/silk-fibroin biocomposite exhibits microscalar confinement of cells and induces anisotropic morphology and migration of embedded fibroblasts.

Microstructural anisotropy of tumor-associated matrix correlates with invasion of cancer cells into the surrounding matrix during metastasis. Here, we report the fabrication and characterization of a three-dimensional (3D) silk-fibroin/collagen-I bio-composite based cell-culture model that exhibits microstructural and biochemical anisotropy. Using RGD-deficient silk-fibroin fibers to confine collagen-I gelation, we develop a silk-fibroin/collagen-I (SFC) bio-composite in a one-step process allowing control over the microstructural and biochemical anisotropy and the pore-size. Two forms of the SFC bio-composite are reported: a sandwich (Sfc ) configuration amenable to live-cell microscopy and an unsupported membrane (Mfc ) for use as a scaffold. Both microscalar and macroscalar mechanical properties of the SFC bio-composite are characterized using atomic force microscope (AFM)-based indentation and tensile-testing. We find that the modulus of stiffness of both Sfc and Mfc can be controlled and falls in the physiological range of 5-20 kPa. Furthermore, the modulus of stiffness of Mfc exhibits a ~200% increase in axial direction of microstructure, as compared to lateral direction. This implies a highly anisotropic mechanical stiffness of the microenvironment. Live-cell morphology and migration studies show that both the morphology and the migration of NIH-3 T3 fibroblasts is anisotropic and correlates with microstructural anisotropy. Our results show that SFC bio-composite permits proliferation of cells in both Sfc and Mfc configuration, promotes cell-migration along the major axis of anisotropy and together with morphological and migration data, suggest a potential application of both the composite configurations as a biomimetic scaffold for tissue engineering applications.

Synthesis and viscoelastic characterization of microstructurally aligned Silk fibroin sponges.

Silk fibroin (SF) is a model candidate for use in tissue engineering and regenerative medicine owing to its bio-compatible mechanochemical properties. Despite numerous advances made in the fabrication of various biomimetic substrates using SF, relatively few clinical applications have been designed, primarily due to the lack of complete understanding of its constitutive properties. Here we fabricate microstructurally aligned SF sponge using the unidirectional freezing technique wherein a novel solvent-processing technique involving Acetic acid is employed, which obviates the post-treatment of the sponges to induce their water-stability. Subsequently, we quantify the anisotropic, viscoelastic response of the bulk SF sponge samples by performing a series of mechanical tests under uniaxial compression over a wide range of strain rates. Results for these uniaxial compression tests in the finite strain regime through ramp strain and ramp-relaxation loading histories applied over two orders of strain rate magnitude show that microstructural anisotropy is directly manifested in the bulk viscoelastic solid-like response. Furthermore, the experiments reveal a high degree of volume compressibility of the sponges during deformation, and also evince for their remarkable strain recovery capacity under large compressive strains during strain recovery tests. Finally, in order to predict the bulk viscoelastic material properties of the fabricated and pre-characterized SF sponges, a finite strain kinematics-based, nonlinear, continuum model developed within a thermodynamically-consistent framework in a parallel investigation, was successfully employed to capture the viscoelastic solid-like, transversely isotropic, and compressible response of the sponges macroscopically.